A Splicing Variation in NPRL2 Causing Familial Focal Epilepsy With

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ARTICLE OPEN A splicing variation in NPRL2 causing familial focal epilepsy with variable foci: additional cases and literature review ✉ Jia Zhang1,2, Yajun Shen1,2, Zuozhen Yang3, Fan Yang3, Yang Li1,2,BoYu4, Wanlin Chen4 and Jing Gan1,2

NPRL2 (nitrogen permease regulator like 2) is a component of the GATOR1(GAP activity towards rags complex 1) proteins, which is an inhibitor of the amino acid-sensing branch of the mTORC1 pathway. GATOR1 complex variations were reported to correlate with familial focal epilepsy with variable foci (FFEVF). However, FFEVF caused by NPRL2 variants has not been widely explored. Here, we describe a variant, 339+2T>C, in NPRL2 identiﬁed by trio whole-exome sequencing (WES) in a family. This splicing variant that occurred at the 5′ end of exon 3 was conﬁrmed by minigene assays, which affected alternative splicing and led to exon 3 skipping in NPRL2. Our cases presented multiple seizure types (febrile seizures, infantile spasms, focal seizures, or focal to generalized tonic-clonic seizures). Electroencephalogram (EEG) showed frequent discharges in the left frontal and central regions. A favorable prognosis was achieved in response to vitamin B6 and topiramate when the patient was seven months old. Our study expands the phenotype and genotype spectrum of FFEVF and provides solid diagnostic evidence for FFEVF.

Journal of Human Genetics; https://doi.org/10.1038/s10038-021-00969-z

INTRODUCTION his mother. The variation was confirmed to impact the alternative NPRL2 is a component of the GATOR1 complex, along with splicing of NPRL2, which may be the consequence of neurogenesis nitrogen permease regulator like-3 (NPRL3) and DEPDC5. The dysplasia through disturbing the mTOR signaling pathway. Our NPRL2 protein is expressed in variable regions of the human brain, study expands the phenotype and genotype spectrum of NPRL2. including the frontal, temporal, parietal, and occipital lobes, similar to DEPDC5 [1]. NPRL2 is also known as tumor suppressor candidate 4 [2], which is upregulated in primary prostate cancer MATERIALS AND METHODS tissues [3]. The GATOR1 complex inhibits mechanistic target of Patient rapamycin (mTOR) activation according to the amino acid levels in Informed consent was obtained from the parents and their families. This ambient cells [4]. It changes the nucleotide loading status (GTP or study was approved by the institutional review board of the West China GDP) of the Rag proteins and deactivates them to release mTOR Second University Hospital. The patient’s clinical manifestations, electro- complex 1 (mTORC1) from the lysosome [5, 6]. encephalogram (EEG), brain magnetic resonance imaging (MRI), malformations, investigations of other organs, and gene variations were analyzed. mTORC1 has been described as a possible cause of We also combined the NPRL2 variant-related cases reported previously in epileptogenesis, and increased activity participates in seizure our analysis. Additional phenotype data and genetic findings for progression [7, 8]. Overall, germline variants in the GATOR1 individuals are summarized in Table 1. complex genes (DEPDC5, NPRL3,andNPRL2) are present in ~10% of focal epilepsy cases [9], which can be familial or sporadic, Whole-exome sequencing (WES) and Sanger sequencing especially in familial focal epilepsy with variable foci (FFEVF). To further clarify the patient’s diagnosis, genomic DNA was extracted from the Moreover, generalized epilepsy and infantile spasms have also peripheral blood of the patient and his parents. WES was performed based on been reported [10]. Loss-of-function variants in DEPDC5 and the NovaSeq 6000 Sequencing platform, IDT XGen Exome Research Panel was NPRL3 have been investigated extensivelyinbothanimal used to capture libraries, and paired-end clean reads were used to compare to models and human tissues associated with mTORC1 hyperacti- the human reference genome (GRCh38/hg38). Variations were annotated vation [11, 12]. However, variants in NPRL2 related to epilepto- through ANNOVAR [14] and picked up with a minor allele frequency of ≤0.005 genesis are still not well understood. in the SNP database. Limited studies have suggested that variants of NPRL2 are WES uncovered potential pathogenic variants. All variants were correlated with focal epilepsy, which may be associated with focal evaluated according to the American College of Medical Genetics and Genomics (ACMG) guidelines. “Ada” and “RF” scores were used to evaluate cortical dysplasia (FCD) and intellectual disability [1, 10, 13]. Our + potential splicing variants predicted by dbscSNV. Sanger sequencing was study uncovered a new variation (NM_006545.5, c.339 2T>C) in performed to validate the variation identified by WES. NLPR2 in a 7-month-old infant with FFEVF that was inherited from

1Department of Pediatrics, West China Second University Hospital, Sichuan University, Chengdu, China. 2Key Laboratory of Obstetrics & Gynecologic and Pediatric Diseases and Birth Defects of the Ministry of Education, Sichuan University, Chengdu, Sichuan, China. 3Cipher Gene LLC, Beijing, China. 4Department of Pediatrics, The City Central Hospital of ✉ Wanyuan, Wanyuan, Sichuan, China. email: [email protected] Received: 26 May 2021 Revised: 23 July 2021 Accepted: 26 July 2021 1234567890();,: 2

Table 1. NPRL2 variations and associated phenotypes in individuals with focal epilepsy identiﬁed to the present.

Case Variation Reference Alternative cDNA Protein Variant GnomAD Novel Gender Age Epilepsy EEG MRI Neuropsychiatric SUDEP Familial Inheritance Penetrance Reference PIMD No. No. allele allele variant alteration class allele classiﬁcation onset phenotype comorbidities in the /Sporadic count family 1 1 G C c.1134C>G p.Cys378Trp Missense 0 VUS Male 3y Frontal lobe Interictal: right FCD (left N/A No N/A N/A N/A Baldassari 30093711 8m epilepsy frontal parieto- et al. [10] epileptiform temporal) activity. Ictal (subclinical): right fronto- central sharp waves, activated by sleep 2 2 G A c.883C>T p.Arg295* LoF 0 Pathogenic N/A N/A Temporal lobe N/A N/A No No Familial N/A Incomplete Ricos 26505888 epilepsy et al. [1] 3 3 C G c.683+1G>C p.(?) LoF 0 Pathogenic Female <1w Infantile Interictal: FCD (left Intellectual No Sporadic N/A N/A Baldassari 30093711 spasms multifocal parieto- disability et al. [10] epileptiform temporal) abnormalities with left predominance 4 4 C G c.640G>C p.Asp214His Missense 22 VUS N/A N/A Frontal lobe N/A N/A No No Sporadic N/A N/A Ricos 26505888 epilepsy, et al. [1] tumor-like brain lesion 5 5 G C c.329C>G p.Thr110Ser Missense 0 VUS N/A N/A Temporal lobe N/A N/A Intellectual No Sporadic Inherited Incomplete Ricos 26505888

epilepsy, disability et al. [1] al. et Zhang J. polymicrogyria 6 6 A G c.314T>C p. Missense 0 Likely N/A N/A Sleep-related N/A N/A No No Familial Inherited Incomplete Ricos 26505888 Leu105Pro pathogenic hypermotor et al. [1] epilepsy 7 6 A G c.314T>C p. Missense N/A Likely N/A N/A Sleep-related N/A N/A N/A N/A N/A Inherited N/A Licchetta 31835056 Leu105Pro pathogenic hypermotor et al. [23] epilepsy 8 7 G A c.232C>T p.Arg78Cys Missense 0 Likely Female 18y Left temporal Left temporal normal No No Familial Inherited Incomplete Perucca 28199897 pathogenic lobe epilepsy slow and spike et al. [24] waves 9 8 G A c.100C>T p.Arg34* LoF 0 Pathogenic N/A N/A Sleep-related N/A N/A No No Familial N/A Complete Ricos et al. 26505888 hypermotor [1] epilepsy 10 8 G A c.100C>T p.Arg34* LoF 0 Pathogenic Female 11m Sleep-related Interictal: FCD (right Intellectual No Familial Inherited N/A Baldassari 30093711 hypermotor discharges in frontal) disability et al. [10] epilepsy right cingulate gyrus 11 9 TGA T c.68_69delTC p. LoF N/A Pathogenic Female 3y Frontal lobe SEEG: right Normal N/A N/A Familial Inherited N/A Weckhuysen 27173016 Ile23Asnfs*6 epilepsy frontoinsular et al. [13] and frontoorbital onset 12 9 TGA T c.68_69delTC p. LoF 0 Pathogenic Female 13y Temporal lobe Left temporal Normal No Yes Familial Inherited Incomplete Weckhuysen 27173016 Ile23Asnfs*6 epilepsy discharges et al. [13] 13 10 C T c.562C>T p.Gln188* LoF/ N/A Pathogenic Male 2y N/A N/A Left superior N/A N/A N/A Inherited N/A Alissa 29281825 Nonsense frontal gyrus et al. [25] FCD 14 11 G A c289G>A, p. Ala97Thr Missense N/A VUS Male 8m Temporal lobe N/A N/A No N/A Familial Inherited N/A Deng 31594065 epilepsy et al. [26] 15 12 T C c.399+2T>C p.(?) Splicing 0 Likely Male 4d focal onset or Frequent Hypoperfusion Moderate No Familial Inherited Complete Proband in our case

ora fHmnGenetics Human of Journal pathogenic focal to discharges in in the cortex of developmental our study generalized left frontal and the left frontal delay tonic-clonic central regions and parietal seizures; regions Spastic Seizures 16 12 T C c.399+2T>C p.(?) Splicing 0 Likely Female 2y Febrile Occasional Relatively Intellectual No Familial Inherited Complete Proband’s our case pathogenic Seizures discharges in hypoperfusion disability mother in the left frontal in the left our study regions frontal and temporal lobes J. Zhang et al. 3 Minigene construction signal abnormalities (Supplementary Fig. 1a–c). Interictal arterial Minigene assays were performed to investigate NPRL2 splicing via WT spin labeling (ASL)-MRI showed relative hypoperfusion in the genomic DNA amplification. The construction contained exon 2–exon cortex of the left frontal and parietal regions (Supplementary 3–exon 4 in NPRL2. Nested PCR was performed to amplify the targeted Fig. 1g–j). His mother had an intellectual disability (WISC Scale fi DNA fragment through normal peripheral blood. Ampli cation products score of 53). Her head MRI was normal (Supplementary Fig. 1d–f), were successfully cloned and confirmed by Sanger sequencing. Variation + NPRL2 while her ASL-MRI showed relative hypoperfusion in the left c.339 2T>C in was constructed by site-directed mutagenesis. Both – wild-type and mutant fragments were delivered into the pcDNA3.1 vector frontal and temporal lobes (Supplementary Fig. 1k n). Her EEG after digestion and connection. All primers used in the minigene construct showed occasional sharp and slow waves in the left frontal are provided in Supplementary Table 1. regions (Supplementary Fig. 2e, f). Both the patient’s mother and grandfather had a history of febrile seizures in childhood.

Cell transfection fi NPRL2 HeLa and 293 cells were cultured in DMEM supplemented with 10% fetal Identi cation of variation related to family focal bovine serum. These minigenes, which are named pcDNA3.1-NPRL2-WT/ epilepsy with variable foci MUT, were transfected into 293T cells using Lipo2000 Transfection Reagent WES was performed to further clarify the diagnosis and explore (11668019, 205 Invitrogen) according to the manufacturer’s protocol. The the etiology of our patient. A heterozygous variant (NM_006545.5: DNA-lipid complex was incubated for 15 min in Opti MEM medium (Gibco, exon 3: c.339+2T>C) in the NPRL2 gene was uncovered, and it was Grand Island, NY) at room temperature before addition to the cells. inherited from his mother (heterozygous) as determined through Sanger sequencing (Fig. 1a, b). The variant was a canonical RT-PCR splicing site in the 5′ end of intron 3, which may impact alternative Total RNA was extracted 48 h after transfection using RNAiso PLUS (9109, splicing. TaKaRa). Retrotranscription was performed using a Prime Script RT Reagent Neither the GnomAD nor Exome Aggregation Consortium kit with gDNA Eraser (RR047A, TaKaRa). Primers are shown in Supplemen- (ExAC) databases exhibited this variation, indicating the rarity of tary Table 1. PCR was performed and evaluated on a 1% agarose gel. this mutation. It was also predicted to be splicing-influencing by fi Subsequently, potential changes in the splicing process were identi ed by “Ada” and “RF” scores (Table 2). Only 27 variants of NPRL2 were direct sequencing. included in the ClinVar database (https://www.ncbi.nlm.nih.gov/ clinvar/?term=NPRL2%5Bgene%5D), 16 of which were pathogenic 3D protein structure modeling or likely pathogenic. To date, 11 variants in the NPRL2 gene have Molecular modeling analysis was performed to estimate the variant in been described in 14 epilepsy probands (Fig. 1c, Table 1). Fifty protein structure. WT and variations in NPRL2 protein were predicted using percent (7/14) of them are pathogenic, 21.4% (3/14) are likely the Swiss-Model program. Swiss-Pdb Viewer software was used to visualize fi the structures between WT and variation proteins. pathogenic, and 28.6% (4/14) are variants of unknown signi cance (VUS). Five variants have been reported to have loss of function (LoF) and were classified as pathogenic in unrelated cases. The + RESULTS c.339 2T>C mutation in our study was also designated as likely Case presentation pathogenic by the ACMG guidelines. A 7-month-old male infant was born after an uneventful full-term pregnancy. He had multiple unprovoked seizures 4 days after Functional splicing examination of the variant through birth, predominantly focal seizures or focal to generalized minigene assays – Since the c.339+2T>C variant changed the 5′ donor site ‘gt’ in the tonic clonic seizures in semiology. The seizures occurred six times fi a day, most of which lasted 10 s and exhibited no diurnal intron region, a splicing assay was performed to further con rm the influence of alternative splicing. Target DNA fragments were differences. He developed spastic seizures when he was 1 month fi old. He was unable to roll over upon admission at 4 months of age successfully inserted into the pcDNA3.1 vector and con rmed by Sanger sequencing (Fig. 2a). RT-PCR results indicated two different with generalized hypotonia evident on the right side. There were “ ” “ ” “ ” “ ” no neurocutaneous markers, specific facial features, or any other splicing patterns, named a and c and b and d (Fig. 2b). Sanger sequencing uncovered abnormal splicing in products “b” systemic abnormalities. His occipitofrontal head circumference “ ” was 42.5 cm. A diagnosis of developmental epileptic encephalo- and d . Intron 2 retention and exon 3 skipping of 18 bp were pathy was considered. Interictal EEG demonstrated frequent sharp observed in the mutant group (Fig. 2c, d). The schematic for abnormal splicing is shown in Fig. 2d. The red asterisk indicates and slow waves in the left frontal and central regions NPRL2 (Supplementary Fig. 2a). Ictal EEG demonstrated the typical the variation site. In addition, exon 3 in is highly conserved findings of epileptic spasms (Supplementary Fig. 2b–d). Brain CT, across multiple species (Fig. 2e). blood glucose, and electrolyte levels were normal. There were no abnormalities in the initial screening for infection or in the blood Protein modeling Swiss-Pdb Viewer was used to predict and compare the structures and urine metabolic screening. In addition, the auditory brainstem + response and visual evoked potential tests were negative. His of NPRL2 in the WT and variant. Variation 339 2T>C causes exon seizures were effectively controlled by a large dose of vitamin B6 3 skipping, which results in several amino acid stretches being (50 mg/kg/d*2 weeks) and topiramate (8 mg/kg/d). Since the deleted. Two sheets and one helix for NPRL2 in the N-terminal seizures free were acquired, the parents refused the treatment of region disappeared compared with the WT (Fig. 3). These regions were highlighted with amino acid residues and van der Waals dots adrenocorticotropic hormone. However, he still exhibited a failure fi to thrive, and his development was delayed and stagnant. At his and will signi cantly affect the function and stability of the NPRL2 latest follow-up at 7 months of age, he was seizure-free and protein. sustained on a combination of topiramate (8 mg/kg/d) and vitamin B6 (10 mg bid) therapy. He had moderate developmental delay, with a Gesell Developmental Scale score of 45. He could lift DISCUSSION his neck at 3 months of age and roll over at 5 months of age. mTORC1 is well known to be involved in cell differentiation and However, he will not sit alone or take the initiative to grasp at protein synthesis and is highly expressed in the brain and 7 months old. Repeated EEG monitoring showed improvement in regulates neurogenesis. Defects in the mTOR pathway play an epileptiform discharge with intermittent sharp and slow waves in important role in focal epilepsy during brain development. the left frontal and central regions. Head MRI showed no obvious GATOR1, a negative regulator of mTORC1, consists of components DEPDC5, NPRL2, and NPRL3. In recent years, GATOR1-related

Journal of Human Genetics J. Zhang et al. 4 c.399+2T>C (a) (b)

Father (T/T)

Mother (T/C) P Febrile Seizures Intellectual Disability Spastic Seizures Focal onset or focal to generalized Proband (T/C) tonic-clonic Seizures

NH2 Longin TINI CTD COOH

p.Cys378Trp VUS p. Ala97Thr p.Asp214His p.Thr110Ser

LP p.Arg78Cys p.Leu105Pro c.399+2T>C Fig. 1 Identification of a heterozygous variation in NPRL2. a Pedigree of the family. The proband affected with focal epilepsy is indicated by filled symbols with arrows. b Sanger sequencing of the proband and his parents showed a c.339+2T>C mutation (red translucent box) in the NPRL2 gene, which was inherited from the mother. c Domain structure and modeling of NPRL2 variations in previous studies. The upper panel indicates the pathogenic (P) variants (red font). Variants of uncertain significance (VUS) and likely pathogenic (LP) are presented below. The NPRL2 protein contains an N-terminal longin domain (NLD), a tiny intermediary of NPRL2 that interacts with the DEPDC5 domain (TINI), and a C-terminal domain (CTD). The variation in our study in the red box was LP.

Table 2. Clinical examinations and variant information. Gene Mutation Inheritance MAF dbscSNV_ dbscSNV_ Category AD_SCORE RF_SCORE ExAc GnomAD 1000 Genome NPRL2 c.339+2T>C Inherited NE NE NE 0.9995 0.866 LP Transcript: NM_006545.5. MAF minor allele frequency, NE not exist, LP likely pathogenic.

variations have been gradually recognized in epilepsy patients via Most patients present with normal psychomotor development, the wide application of second-generation sequencing, causing while some may have mild cognitive decline or autism-like ~10% of focal epilepsy [1, 13]. GATOR1 variations that activate the manifestations without seizures [1, 15]. Similarly, our case mTORC1 pathway assume the main responsibility for focal presented multiple seizure types, including infantile spasms, epilepsy with cortical malformations, representing a potential focal seizures, or focal to generalized tonic-clonic seizures. Both target for novel therapeutics [10]. his mother and grandfather had a history of febrile seizures Familial focal epilepsy (FFE) is characterized as a genetically when they were young. In addition, his mother had an distinct type of epilepsy syndrome, primarily including auto- intellectual disability. somal dominant nocturnal frontal lobe epilepsy, familial medial Approximately 20% of patients with GATOR1-related focal temporal lobe epilepsy, familial lateral temporal lobe epilepsy, epilepsy have FCD [10, 13, 16]. In our study, no obvious and FFEVF, which are considered to primarily result from ion abnormalities were observed in MRI of this proband. ASL-MRI channel and neurotransmitter receptor gene variations. FFEVF is showed relative hypoperfusion in the left frontal and parietal a typical subtype that has been reported to be primarily lobes, consistent with the epileptiform discharge detected by associated with DEPDC5 variations. At the same time, ~12% of video EEG. It has been reported that 55–78% of GATOR1-related FFE patients have been found possess DEPDC5 variations [15]. focal epilepsy patients are drug-resistant [10, 17]. Patients with Epilepsy syndrome caused by NPRL2 and NPLR3 variations is deﬁnite lesions had better surgical results, with a 50–60% similar to DEPDC5 [1]. EEG showed that the initial discharge site complete remission rate [10, 18]. Fortunately, our case had a was changeable, mostly from the frontal lobe or temporal lobe. favorable prognosis with the combination treatment of topir- However, no obvious correlation between EEG discharge and amate and vitamin B6, and the video EEG on follow-up showed a clinical manifestations was found [16]. GATOR1-related FFEVF signiﬁcant decrease in epileptiform discharges. Considering the patients always have a family history, with incomplete focal seizures with epileptic spasms of the proband, high-dose penetrance and heterogeneous clinical manifestations. Some vitamin B6 and topiramate were applied according to the of them exhibit focal EEG discharge without clinical symptoms. guidelines and consensus on the treatment of infantile spasms

Journal of Human Genetics J. Zhang et al. 5

Fig. 2 Minigene assays and identiﬁcation of the variant’s impact on alternative splicing. a Sanger sequencing conﬁrmed that wild-type and mutant fragments were successfully introduced into the minigene construct. Splicing variation c.339+2T>C in NPRL2 is indicated by the red box. b RT-PCR was performed to verify alternative splicing in the wild-type and mutant groups. Abnormal splicing bands in mutant groups, named “b” and “d”, were uncovered in both HeLa and 293 T cells. At the same time, normal bands, named “a” and “c”, were indicated in the wild-type group. c Alternative splicing was affected by the c.339+2T>C variation in NPRL2. PCR product sequencing revealed 18 bp intron 2 retention and exon 3 skipping. The alternative schematic is shown in (d). The red “*” symbolizes the variation site. Intron 2 retention is indicated in the mutant group by a red line. e Species conservation analysis of exon 3 in NPRL2.

There is no clear correlation between genotype and phenotype in (a) GATOR1 variations, with an incomplete penetrance from 50% to 82% [1, 15]. This likely results from a combination of genetic, environmental, and lifestyle factors [20]. Additionally, this may be due to the additive effect of multiple independent variations, which can disturb and often increase the severity of the phenotype. GATOR1-related epilepsies caused by DEPDC5, NPRL2,andNPRL3 N terminal variations exhibit various phenotypes consisting of sleep-related focal hypermotor seizures, infantile spasms, and other focal C terminal epilepsies, including frontal, temporal, occipital, parietal, centrotem- poral epilepsies [10]. The various phenotypes are the same as those of patients who have NPRL2 variations. Previous studies also revealed distinct phenotypes in a family with the same variant (b) [1, 13]. A single pathogenic variant could lead to variable phenotypic manifestations, including different age at onset, seizure type, seizure severity, drug response, and presence of cortical malformations [16], which was observed in our comparison (Table 1). The types of variations seem to have no connection with the epilepsy phenotypes, neuropsychiatric comorbidities, or abnormal brain structure. Interfamilial variability in genetic epilepsies is a common N terminal phenomenon. Therefore, the child presented with developmental epileptic encephalopathy with focal as well as spastic seizures, while C terminal the mother showed only mild mental retardation. The maternal Fig. 3 Protein 3D structures in WT and the NPRL2 variant. a WT grandfather had a history of febrile seizures at a young age, and protein. b Variant protein. The yellow dotted box indicates the then had no epileptic seizures, and the psychomotor development different regions of the WT NPRL2 protein compared to the variation. was normal. One explanation for how a single variant in the same family causes mild focal epilepsy or refractory epilepsy may be the and epileptic encephalopathy [19]. However, there are not any occurrence of a second somatic mutation [18]. apparent relationships between the NPRL2 gene and the Haploinsufﬁciency has been shown to be the pathogenic mechanisms of action of anti-seizure medications which needs mechanism in NPRL2/3 variations with incomplete penetrance more investigation in the future. [13]. However, whether missense and splicing variations have

Journal of Human Genetics J. Zhang et al. 6 clinical significance is still unclear. Functional evidence and strong 18. Baulac S, Ishida S, Marsan E, Miquel C, Biraben A, Nguyen DK, et al. Familial focal segregation support were absent. Therefore, no missense or epilepsy with focal cortical dysplasia due to DEPDC5 mutations. Ann Neurol. splicing variations were classified as pathogenic through the new 2015;77:675–83. framework of epilepsy-related GATOR1 classification [10]. Alter- 19. Wilmshurst JM, Gaillard WD, Vinayan KP, Tsuchida TN, Plouin P, Van Bogaert P, native splicing was confirmed by minigene assays, and exon et al. Summary of recommendations for the management of infantile seizures: 3 skipping was observed during transcription (Fig. 2). The Task Force Report for the ILAE Commission of Pediatrics. Epilepsia. 2015;56:1185–97. mutation may destroy the original donor site in exon 3 and 20. 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